Structural insights into the activation of ataxia-telangiectasia mutated by oxidative stress

Ataxia-telangiectasia mutated (ATM) is a master kinase regulating DNA damage response that is activated by DNA double-strand breaks. However, ATM is also directly activated by reactive oxygen species, but how oxidative activation is achieved remains unknown. We determined the cryo-EM structure of an H2O2-activated ATM and showed that under oxidizing conditions, ATM formed an intramolecular disulfide bridge between two protomers that are rotated relative to each other when compared to the basal state. This rotation is accompanied by release of the substrate-blocking PRD region and twisting of the N-lobe relative to the C-lobe, which greatly optimizes catalysis. This active site remodeling enabled us to capture a substrate (p53) bound to the enzyme. This provides the first structural insights into how ATM is activated during oxidative stress.

that interact with the activation loop, catalytic loop, LBE and p53 peptide, as shown in the H2O2activated state (left) where one protomer is represented with ribbons and the other protomer is represented with cylinders.Deletion of these residues in the H2O2-activated ATM dimer could destabilize the activation loop, catalytic loop and LBE, preventing an optimal conformation in the active site for catalysis and p53 substrate binding.ATM is hypothesized to form a monomer in the MRN/DNA activating condition, and so a single protomer derived from the H2O2-activated structure is used to represent this (right).MRN/DNA could potentially form additional stabilizing interactions with the LBE and activation loop that could not be provided by the other ATM protomer in the H2O2-activated ATM dimer.
Table S2.Features and residues involved at the upper dimeric interface between ATM protomers.Features and residues that are involved at the upper dimeric interface are shown and juxtaposed for the ATM basal state (left) and ATM H2O2-activated state (right).Residues that have a buried residue area of at least 15 Å are considered to be interface residues and are listed in brackets below the associated feature.Table S3.Plasmids used for protein expression.
Movie S1.The ATM dimer is twisted in the ATM H2O2-activated state compared to the basal state.ATM protomer rotation is observed when morphing the basal state model and H2O2-activated state model.Models are aligned on the right-hand side protomer (gray or dark pink) to observe changes occurring in the left-hand side protomer (white or light pink).
Movie S2.An additional bridge density is observed between the two ATM protomers in the H2O2-activated state consensus map.An additional bridging density is observed in the 7 Å lowpass-filtered H2O2-activated state ATM consensus map.The grey map (consensus map) has its density threshold lowered whilst the pink map (the high resolution 3 Å sharpened C terminus dimer map) remains at a higher density threshold and is used as a reference to show the relative position of the bridge between ATM protomers.The p53 substrate peptide bound in the active site is shown as magenta sticks.Side chains are shown for the C-lobe residues that make up the pocket into which the p53 Q16 (position +1) side chain inserts.In the activated state, helix kα9b of the PRD is mostly disordered and not seen in the structure.For the movie, helix kα9b, which is ordered and wedged between the N-and Clobes in the basal state, is shown lifting out of the active site upon activation and assuming an arbitrary position, while the p53 substrate peptide slides into the emptied active site.The side chain for Q2971 in helix kα9b is illustrated as sticks to emphasize that in the basal state Q2971 occupies the same pocket as Q16 of the substrate peptide in the activated state.Upon activation, the N-lobe of the kinase domain (red) rotates relative to the C-lobe (yellow).This causes the bound ATP analogue (cyan, AMPPNP) to change its conformation, altering interactions with the bound Mg 2+ (large cyan sphere), and changing ATP contacts with the N-lobe.Once the p53 substrate is in place, the g-phosphate of the ATP analogue (light blue sphere) forms a close interaction with S15 of the substrate peptide.

Fig. S1 .
Fig. S1.Domain organization of p53 substrate and the structure of ATM kinase.The human p53 peptide sequence (residues 11-22, within the transactivation domain (TAD1) used in our cryo-EM sample is highlighted on a bar diagram showing the p53 transactivation domains (TAD1 and TAD2), DNA-binding domain (DBD), tetramerization domain (TD) and C-terminal domain (CTD), The domain architecture and structure of ATM (PDB: 7SIC) illustrate the locations of the N-solenoid, Pincer, FAT, kinase domain and FATC.Inset: Close-up image of the PRD kα9b helix.

Fig. S5 .
Fig. S5.Cryo-EM map quality metrics for the ATM basal state and H2O2-activated state dimer C-terminus maps.(A) Front and side views of sharpened EM density maps colored by local resolution, estimated using cryoSPARC v4.(B) Euler angle distribution for particles contributing to the final 3D reconstructions.EM density maps are shown separately to the right for clearer illustration of the map orientation.(C) Directional FSC plots and histograms for the final reconstructions calculated using 3DFSC (61).

Fig. S6 .
Fig. S6.Cryo-EM map quality metrics for the ATM H2O2-activated state and basal state dimer consensus maps.Front and side views of unsharpened EM density maps (left) for (A) ATM H2O2-activated state and (B) basal state colored by local resolution, estimated using cryoSPARC v4.Directional FSC plots and histograms for the final reconstructions were calculated using 3DFSC (right).

Fig. S7 .
Fig. S7.ATP loop shifts are observed for the activated states of different PIKK family members.Models for basal and activated states of PIKK enzymes are aligned on the catalytic and activation loops in the kinase C-lobes.The activated states show a shift in the ATP loop relative to the basal state.

Fig. S8 .
Fig. S8.Structure-based multiple sequence alignment of PIKKs.H. sapiens ATM, S. cerevisiae ATR (Mec1), H. sapiens mTOR, and H. sapiens DNA-PKcs sequences were aligned for the ATP loop, catalytic loop, and activation loop.The pairwise structure alignment tool from the PDB was first used on 7SIC (human ATM) paired with either 6Z2X (yeast ATR), 6BCX (human mTOR), or 7K11 (human DNA-PKcs).Sequence alignment was then performed for each pair for more extensive sequence coverage (to cover regions not included in PDB models) using MAFFT version 7 (62).Uniprot sequences Q13315 (human ATM), P38111 (yeast ATR), P42345 (human mTOR), and P78527 (human DNA-PKcs) were used for the sequence inputs, and the pairwise structure alignment fasta file was used for additional constraints.Manual adjustments were then made in JalView (version 2.11.2.6) (63) to align the kinase domains.ESPript 3.0 (64).

Fig. S9 .
Fig. S9.Modelling of the disordered PRD regions into the ATM dimeric C-terminal regions.(A) A model for the H2O2-activated state with the addition of a plausible model for the disordered PRD region (left) and the 7 Å lowpass filtered consensus map superimposed on the model (right).(B) Same as in (A), but for the ATM basal state.The disordered PRD regions that cannot be modelled into high-resolution density of the locally refined dimeric C-terminal region maps are colored green and account for the remaining residues within the PRD.C2991 residues are indicated by yellow spheres.

Fig. S11 .
Fig. S11.Interactions of PRD residues L2970 and Q2971 with surrounding residues in basal state ATM.L2970A and Q2971A mutants were generated to destabilize the PRD from the substrate binding site.The interactions of the wild-type L2970 (left) and Q2971 (right) with the surrounding ATM residues are shown using PDB: 7SIC.L2970 makes hydrophobic interactions with residues V2892, L2900 (in the activation loop) and M2962 (in the PRD).Q2971 makes hydrogen bonds with the peptide backbone of T2902 (in the activation loop) and hydrophobic interactions with L2900 (in the activation loop) and F3049 (in FATC).

Fig. S12 .
Fig. S12.ATM basal state cryo-EM maps.(A) (i) Local refinement maps for the N-terminus (two copies) and dimeric C-terminal region are docked into the whole dimer consensus map.(ii) The ATM basal state whole dimer model.(B) Processing steps for local refinement of the Nsolenoid and pincer region.(C) Close-up view of the additional PRD linker density and FLAP-BE' tip density in the basal state map.The contact of these densities indicates potential interactions between PRD linker and FLAP-BE'.

Fig. S13 .
Fig. S13.ATP hydrolysis by ATM, using ADP-Glo kinase assay.ATP hydrolysis rates were measured for samples containing ATM without activator (basal), ATM with H2O2 and ATM Δkα9b mutant.Data were obtained from three biological replicates.Averages are plotted with error bars showing standard deviations.Data values are displayed in a table below the graph.

Fig. S14 .
Fig. S14.Comparing the effects of R3047X on H2O2-activated ATM versus MRN/DNAactivated ATM.Mutation R3047X deletes the last ten residues in FATC (highlighted in black)that interact with the activation loop, catalytic loop, LBE and p53 peptide, as shown in the H2O2activated state (left) where one protomer is represented with ribbons and the other protomer is represented with cylinders.Deletion of these residues in the H2O2-activated ATM dimer could destabilize the activation loop, catalytic loop and LBE, preventing an optimal conformation in the active site for catalysis and p53 substrate binding.ATM is hypothesized to form a monomer in the MRN/DNA activating condition, and so a single protomer derived from the H2O2-activated structure is used to represent this (right).MRN/DNA could potentially form additional stabilizing interactions with the LBE and activation loop that could not be provided by the other ATM protomer in the H2O2-activated ATM dimer.
-TEV-NRF1 (full-length)AHp46pOPTHM(TEV) 6xHis-MBP-TEV-CHK2(1-107) S3.The upper dimeric interface changes in the H2O2-activated state compared to the basal state.Dimeric interface interactions change going from the basal state to the H2O2activated state of ATM.The movie begins with the ATM dimeric C-terminal region, then focuses in on the dimeric interface on one side of the dimer.Elements involved at the dimeric interface are colored and then morphing occurs from the basal state to the H2O2-activated state.Movie S4.The kinase N-lobe rotates relative to the C-lobe in the H2O2-activated state compared to the basal state.A morph movie demonstrating the rotation of the kinase N-lobe (white or light pink) relative to the C-lobe (gray or deep pink) going from the basal state model (white and gray) to the H2O2-activated state model (light and deep pink).The models are aligned on the kinase C-lobes to focus on motion in the N-lobe.Movie S5.Remodeling of the kinase domain upon H2O2-mediated activation.A morph movie of the kinase domain going from H2O2-activated ATM with p53 substrate bound to the basal state (PDB: 7SIC).Similarly, the bound ATP analogue of the activated state was morphed onto the ATP analogue of the basal state.Only the kinase domain and its substrates are shown.